PROJECT SUMMARY While circulating endothelial cells (ECs) show the potential to home to sites of vascular injury, aiding in its repair, they are difficult to isolate and expand in vitro, requiring animal products that are potential pathogenic, curtailing their use as vascular therapeutics. To this end, human pluripotent stem cells (hPSCs) including human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCS), serve as a potential renewal source of ECs. While their derivation from hPSCs have been achieved, the chemical cues used to induce their differentiation is ill-defined due to the inherent variability in animal serums used in the maturation media. In addition, the effect of physical cues relevant to the developing vasculature such as confinement and shear stress towards EC fate decision and arterial/venous specification have yet to be elucidated. The goal of this proposed research is to achieve a homogenous population of arterial or venous ECs in a clinically relevant manner. Our aims are to: (1) Establish a chemically-defined differentiation protocol that promotes EC commitment from hPSCs; (2) guide EC fate via confinement in chemically-defined culture conditions and (3) determine the effect of shear stress on EC arterial/venous specification. To achieve these aims, a highly interdisciplinary approach is taken, incorporating the fields of stem cell and vascular biology as well as engineering principles. Successful completion of these aims will broaden our understanding of the physio-chemical factors driving stem cell fate, with considerable impact in using the differentiated cells as vascular therapeutics.